Gene Mutations

The Place of Genetics in

Medicine
• The true scale of genetic disease has
only recently appreciated.
• One in 20 people under the age of 25
develop a serious disease with a
major genetic component.
• Studies of causes of death of more
than 1200 British children suggest
that 40% died as a result of a genetic
condition.
Mutations
• Some mutation is good, too much is
bad.
• Cells employ elaborate mechanisms to
prevent mutation, but the mechanism
aren’t perfect.
• Mutations are the root cause of cancer
(bad)
• Mutations are the only way to introduce
novel genes into a species for evolution
(good).
• The effects of mutation are usually bad
or neutral - only sometimes are
mutations beneficial.
• So just like Goldilocks - not too hot, not
Gene Mutations &
Diseases
• Many, if not most, diseases have
their roots in our genes.
• More than 4000 diseases stem from
altered genes inherited from one’s
mother &/or father.
• Common disorders such as heart
disease & cancer arise from a
complex interplay among multiple
genes & between genes and factors
in the environment
Gene Mutations
• Genes can be altered or mutated in
many ways
• The most common gene change
involves a single mismatch
(misspelling) placing the wrong base
in the DNA
• At other times, a single base may be
added or dropped
• And sometimes, large pieces of DNA
are mistakenly repeated or deleted
Classification of Mutations
• Single base substitution (also called
point mutations)
– Missense mutation (sickle cell
disease)
– Nonsense mutation (cystic fibrosis)
– Silent mutation (one in G6PD
deficiency)
– Splice-site mutation (thalassemia)
• Insertion & deletions
Mutation Definition

Transversion

Pu to Pu - transion
uu u u
Generation of a 'nonsense'
mutation
•  The wild-type DNA sequence 
   5'- CTA CAG ATT - 3'
                                   3'- GAT GTC TAA - 5'
• Produces the mRNA 5'- CUA CAG AUU - 3'
 which codes for the polypeptide 
leu gln iso [etc]
• A 1st position mutation (C T) in the second
triplet gives a 'mutant' DNA sequence
   sense strand           5'- CTA TAG ATT
- 3'
                          3'- GAT ATC TAA
- 5'
Produces the mRNA which 5'- CUA UAG
AUU - 3'
codes for the polypeptide leu- *
Generation of an 'amber suppressor'
mutation in a tRNA gene
• DNA sense strand   5'- TAC -3'
                            3'- ATG -5'
   which produces the tRNA   3'- AUG -5'
anticodon loop
   which reads the mRNA      5'- UAC -3'
   as                             - tyr –

•   A 3rd-position mutation (C- G) in this region

produces a 'mutant' tDNA gene

•   sense strand               5'-TAG -3'

                             3'- ATC -5'
  which produces the tRNA    5'- AUC -3' 
anticodon loop
  which reads the mRNA as  5'- UAG -3'
                         - tyr -
Insertions & Deletions
• Each base pairs may be
added(insertions) or removed (deletions)
from the DNA of a gene
• Insertions & deletions involving one or
two base pairs( or multiple thereof)) can
have a devastating consequences to the
gene because translation of the gene is “
frameshifted”
• Insertions or deletions of three
nucleotides or multiple of three may be
less serious because they preserve the
reading frame
• A number of inherited diseases are
caused by the insertion of many copies
Duplications
• Duplications are a doubling of a section of
the genome during meiosis
• This can cause the new gene to carry
inappropriate promoters at its 5’ end
Translocation
• Transfer of a piece of one chromosome to
a nonhomologous chromosome
• This can alter the phenotype in several
ways
• Break may occur within a gene destroying its function
• Gene may come under influence of different
promoters & enhancers
• Breakpoint may occur within a gene creating a
 mRNA processing
Normal splicing
defects
EXON 1 EXON 2 EXON 3

EXON 1 EXON 2 EXON 3

CACCC CCAAT IVS I IVS II
ATAA

Abnormal splicing
EXON 1 EXON 2 EXON 3

β + thalassemia IVSI-
Frequency of Mutations
• Mutations are rare events.
• Humans inherit 3 x 109 bp from each parent. i.e.
each cell has 6 billion different bp that can be the
target of a substitution.
• Substitution occurs when DNA is being copied
(during S phase).
• No process is 100% accurate. So it is with DNA
replication.
• Errors occur at a rate of about 1 in every 50
million nucleotides added to the chain. (Not bad, I
wish I could type so accurately.)
• But with 6 X 109 bp in a human cell, it means
that each new cell contains some 120 new
mutations.
• Should we be worried? Probably not.
• Most our DNA does not encode anything. This
includes:
– Repetitive DNA(Alu-junk) –introns –synonymous codons
Somatic vs Germline
Mutations
• Mutations that occur in a somatic cell,
bone marrow or liver, may
•Damage the cell
•Make the cell cancerous
•Kill the cell
Whatever the effect, the ultimate fate of
that mutation is to disappear when the cell
in which it occurred , or its owner , dies
• Germline mutations, in contrast, will be
found in every descendant from zygote to
which that mutant gamete contributed. It
will be passed from one generation to the
Males contribute more
mutations than Females
• Most mutations occur during S phase of
cell division. Consequently males
should be more at risk because:
– Only two dozens or so mitotic
divisions occur from the fertilized egg
that starts embryonic developments
– The sperm of a 30 year old man in
contrast is the descendant of at least
400 mitotic divisions since the
fertilized egg that formed him
• The children of aged father suffer more
genetic disorders than those of young
 Altered DNA-Altered
Protein
• A protein encoded by a gene that
contains a mutation is likely to be
abnormal.
• Sometimes the protein will be able to
function, but imperfectly.
• In other cases, it will be totally
disabled.
• The outcome depends not only on
how it alters a protein’s function but
Hereditary Mutations
• Gene mutations can be either
inherited or acquired
• Hereditary mutations are carried
in the DNA of the reproductive
cells
• When productive cells containing
mutations combine to produce
offspring, the mutation will be in
all of the offspring’s body cells
Acquired Mutations
• Acquired mutations are changes in
DNA that develop throughout a
person’s lifetime
• Although mistakes occur in DNA all
the time, a cell has the remarkable
ability to fix them
• But if the DNA repair mechanism
fails, mutations can be passed
along to future copies of the
altered cell
Alleles
• Genes come in pairs, with one copy
inherited from each parent.
• Many genes come in a number of variant
forms, known as alleles.
• A dominant allele prevails over a normal
allele.
• A recessive gene becomes apparent if its
counterpart allele on the other
chromosome becomes inactivated or lost.
• During formation of gametes, the
members of each pair separate, so as each
gamete carries one allele of each pair.
Allele pairs are restored at fertilization.
• Alleles of different genes reassort
Altered Dominant Genes

• In a dominant genetic disorder, one

affected parent has a disease-
causing allele that dominates its
normal counterpart
• Each child in the family has a 50-
percent chance of inheriting the
disease allele and the disorder
Rules for autosomal dominent
inheritance
• Both males & females express the allele
& can transmit it equally to children.
• Excluding new mutations & non-
penetrance, every affected person has
an affected parent.
• Direct transmition through three
generation is practically diagnostic of a
dominant.
• In affected families, the ratio of affected
to non affected is almost always 3 : 1
• If both parents are unaffected all
Brachydactyly
• Abnormally short phalanges.
• Dominant allele B causes brachydactyly &
every affected person is either BB or Bb.
• In practice most heterozygote, because
brachydactyly is a rare trait.
• Estimation of risk for offspring of a family
member can be based simply on the
predictions of Mendel’s laws.
• However calculations of dominant
condition can be problematic since we
usually do not know whether an affected
offspring is homozygous or heterozygous.
Altered Recessive Genes
• Here both parents, though disease free
themselves, carry one normal allele and
one altered allele
• Both males & females are affected
• Each child has one chance in four of
inheriting two altered alleles and
developing the disorder, one chance in
four of inheriting two normal alleles, and
two chances in four of inheriting one
normal and one altered allele and being a
carrier like both parents
• When both parents are affected all the
children are affected
Albinism
• Represent one in 10000 births
• They have very pale hair & skin, blue or
pink irides & red pupils
• Suffer from photophobia, nystagmus(
involuntary eye movements)
• Due to deficiency of tyrosinase.
• Every albino is a recessive homozygote
(aa) and most are born to phenotypically
normal parents.
Disease Inheritance is
Complex
• Most diseases do not follow
simple patterns of inheritance.
• Many factors influence a gene’s
ability to build proteins.
• Different mutations in the same
gene can produce a wide range
of effects.
• Cystic fibrosis is a good example.
Dominance & Codominance
• Dominance is defined in terms of which two
alternative allele is expressed in the
heterozygote.
• In the ABO blood group system, groups A, B, AB
& O are distinguished by whether the RBCs are
agglutinated by anti-A or anti-B.
• RBC of both IA homozygote and IA/IO
heterozygotes are agglutinated by anti-B
antibody (similar in IB & IBIO).
• Therefore allele IA & IB are dominant to IO.
• The RBCs of IO homozygons are not agglutinated
by both antibodies (group O).
• RBCs of group AB individuals carry both A & B
antigens. They are agglutinated by both
antibodies. Since both expressed, alleles IA & IB
are codominant.
Intermediate Inheritance
Incomplete dominance – refers to the
situation when gene expression in
heterozygote is intermediate
between that in the two homozygote.
• Example: sickle cell anemia.
Heterozygous have both HbA & HbS
in their erythrocytes which stay
undistorted most of the time. At this
level HbA is dominant to HbS.
• Under conditions of hypoxia, a
proportion do undergo sickling, on
this basis HbS is classified as
Pleiotropy
• Describes the expression of several
different phenotypic features by a
single allele.
• Most genes have pleiotropic alleles.
• An example is Marfan syndrome (tall,
long, thin limbs & fingers,
susceptibility of aorta to aneurysm
(swelling) due to fibrillin -1.
• Those that have the disease can vary
in severity (variable expressivity)
Sex-Related Inheritance
(Recessive)
• Rules:
– The incidence of disease is very much
higher in males than females.
– Mutant allele is passed from an affected
man to all his daughters, but they do not
express it.
– A heterozygous “carrier” woman passes
the allele to half of her sons, who
express it and half of her daughters who
do not.
– The mutant allele is never passed from
father to son.
– Example is G6PD deficiency.
Sex-Related Inheritance
(Dominant)
• Rules:
– The condition is expressed and
transmitted by both sexes.
– The condition occurs twice as frequently
in females as in males.
– An affected man passed the condition to
every daughter, but never to a son.
– An affected woman passes the condition
to every son and half her daughters.
– Example: Hypophosphataemia
Mitochondrial Inheritance
• Mitochondrial inheritance reflects the fact
that a zygote receives all its functional
mitochondria from the oocyte.
• Rules:
– Typically the condition is passed from a mother
to all her children.
– The condition is never transmitted by men.
– Symptoms can vary between mother and
offspring, and between sibs, due to
heteroplasmy, i.e. variable representation of
different mitochondrial populations.
• Example: A form of progressive blindness
called Leber hereditary optic neuropathy
MITOCHONDRIAL DISORDERS
 Exceptions to Mendelian
•Inheritance
X Inactivation: The Lyon Hypothesis
• One of the two X-chromosomes in the
female is functionally inactivated.
• The inactivation occurs early in the life of
the embryo.
• In the embryonic cell, it is a matter of
chance as to which of the two X-
chromosomes (P or M) is inactivated.
• Once an X-chromosome is inactivated in
an embryonic cell, all the progeny of that
cell maintain the same inactive X.
• In individuals with more than two X-
chromosomes, all those in excess of one
are inactivated.
Female is a Mosaic of X-
chromosome gene expression
:with
• Patches of cell expressing the genes
of the paternally derived X.
• Patches of cell expressing the gene
of the maternally derived X.
• On average, the split will be 50:50
• Considerable variation could, and in
fact does occur because inactivation
event is random.
Clinical Implication of X
.Inactivation
• X-linked recessive disorders.
• In many X-linked recessive disorders,
the heterozygotes have a remarkable
range of expression varying from
being phenotypically normal to
manifesting characteristics of the
disorder almost as severely, or as
severely as the affected male.
• Skewed inactivation (randomness of
X-inactivation)
Chemical Mutagenesis
• Environmental mutagens include
constituents of smoke, paints,
petrochemicals, pesticides, dyes, food
stuffs, drugs etc.
• Principle means of exposure are inhalation,
skin absorption and ingestion.
• Effects include:
– Nitrous acid causes transition (C U)
– Chemicals which cause DNA strand
breakage
– Acridine dyes intercalate between
adjacent base pair and leads to deletion
Electromagnetic Radiation
• Includes α, β and γ radiation.
• Their mutagenicity increases with
decreasing wavelength.
• All radiations beyond UV causes ionization.
Sources include
– Natural background radiation (Potassium 40
and radon 222 gas)
– Cosmic rays, major hazards for aircraft as their
intensity increases with altitude.
– Man-made radiation nuclear testing, power
stations.
– X-rays
Biological Effects of Radiation
((1
• Electromagnetic radiation damages
proteins and above 100 rads kills
cells.
• At chromosomal level it causes major
deletions, translocation and
aneuploidy.
• It causes single and double-strand
breaks in DNA and base pair
destruction.
• X-rays damage chromosomes most
readily when they are condensed,
Biological Effects of Radiation
((2
• For the first seven days, embryo is
ultrasensitive to the mutagenic
and lethal effects of x-rays.
Childhood leukemia can be
induced by exposure at gestational
weeks 8-40.
• 100 rads causes 50% drop in WBC
count, at 450 rads kills 50% of
people, 1000 rads used to kill
Fragile X-Syndrome
Case: A 10 year old male is referred to a
genetic evaluation clinic by his
pediatrician because of mental
retardation.
Labs: All basic & endocrinological lab profiles are normal.
Karyotype “ fragile gap” at the end of the long arm on X-
chromosome.
• Second most common form of inherited
mental retardation.
• Expansion of trinucleotide repeat
sequence CGG found on the
5’-untranslated region of FMR1 gene on X
chromosome ( > 200 repeat).
• This induces extensive methylation of the
promoter region & inactivates it, which
Thalassemia
• Imbalance in β & α globin peptide
chain synthesis.
• Anemia at age of 6 months and
requires frequent transfusion.
• α-thalassemia is usually due to
deletion.
• β-thalassemia is due to a wide variety
of mutations including frame shift,
truncation & splice mutation.
 Death CapMushroom
((amanita phalloides
 This is a highly poisonous mushroom that
causes several deaths each year.
 It contains a lethal toxin α-amanitin that
inhibits the largest subunit of eukaryotic RNA
poly II, thereby inhibiting mRNA synthesis.
 Initial poisoning is relatively mild – such as
gastrointestinal symptoms.
 This is followed 48h later by massive liver
failure as essential mRNAs and their proteins
are degraded but not replaced by newly
synthesized molecules.
A Case of Mistaken Identity((1
An otherwise healthy young man presents
himself to the emergency room with
severe nausea and diarrhea. His
symptoms came on suddenly, about 2-3
hours after he had eaten dinner. The
physician suspects some form of food
poisoning, and asks the patient to recall
everything he has eaten over the past 24
hours. The only suspicious food mentioned
were mushrooms that the patient ate for
dinner. The mushrooms become prime
candidates for the cause of this patient’s
symptoms when he further relates that
they were picked up on a recent hike
through the woods. What is the
biochemical basis for suspecting that the
 A Case of Mistaken Identity((2
Comment: It is likely that the patient
mistakenly picked a member of the family
Amanita phalloides and ingested them at
dinner. The toxin, α-amanitine, binds
preferentially to RNA polymerase II and
inhibits its function; if a large quantity of the
mushrooms had been ingested, even RNA
polymerase III could be inhibited. The first
cells that encounter the toxin are those of the
digestive tract, leading to acute
gastrointestinal distress: cells that are
incapable of synthesizing new mRNAs and
tRNAs would die, causing the diarrhea and
Rifampicin
• Tuberculosis is endemic in
impoverished populations.
• Mucobacterium tuberculosis, the
causative agent of tuberculosis is
insensitive to many commonly used
antibiotics, but it is sensitive to
rifampicin.
• Rifampicin, produced by soil
streptomycetes and is an inhibitor of
many bacterial RNA polymerases.
• Eukaryotic polymerase is not that
sensitive.
Systemic Lupus
Erythematosus
• An autoimmune disease of the
connective tissue.
• Patients exhibit a serum antibody
designated Sm.
• This serum antibody binds to
common core proteins of snRNP (U1,
U2, U4, U5)
• snRNP are involved in spliceosomes
which are responsible for mRNA
processing.
• Hence antibody inhibits splicing.
Hemoglobin
McKees
• Characterized by polycynthemis (over
production of RBC due to increase
secretion of erthropoietin secreted by
the kidneys)
• Position 145 of the β-chain normally
occupied by tyrosine which is coded
for by UAU or UAC.
• Mutations in this codon to UAA or UAG
result in termination of β-globin chain
synthesis.
• This gives rise to hemoglobin
molecules (144 AA instead of 146)
 Southeast Asiaβ-
Thalassmia
 Imbalance between α & β-globin chain
synthesis.
 β -globin is not synthesized; results in
α-globin accumulation and precipitation
in erythroid cells.
 This precipitation damages cell
membrane
and causes hemolytic anemia.
 Normal codon 17 in β-gene (AAG) codes
for lysine, a mutation leads to UAG (stop
codon) which terminates β-globin chain
synthesis.
 The mutation occurs so early in mRNA
that no useful β-globin sequence can be
synthesized.
 Constant Springα
-Thalassmia
• This is characterized by abnormally long
α-globin molecule which is unstable &
breaks easily.
• α-codon 142 (UAA) designates a stop
codon with formation of a normal α-globin
chain with 141 amino acids.
• Mutation to CAA replaces stop codon with
codon codes for glutamine (in the
untranslated region)
• The next stop codon is at 173, hence
production of the α-globin with 172 amino
Antibiotic-Induced Deafness
• In some regions of China, use of gentamicin &
streptomycin leads to deafness.
• This is a maternally transmitted sensitivity which is
therefore due to mitochondrial DNA.
• Codon 1555 of the gene on mitochondrial DNA codes
for the rDNA of the A-site of the large subunit.
• A mutation A to G results in making the region more
prokaryote-like, increasing its affinity for
aminoglycosides binding
• Antibiotc bindings therefore interfere in protein
synthesis in the mitochondria.
• This antibiotic tends to accumulate in the cochlea,
making this a particularly sensitive target and leading
to sensorineural deafness.
Ehlers-Danlos Syndrome
• Case: A 9-year-old boy is brought to ER with pain,
inability to lift his left shoulder and flattening of
the normal rounded shoulder contour (Shoulder
dislocation) that occurred when he tried to hit a
ball with a baseball bat.
• PH: He dislocated his left shoulder nine times
before and his right shoulder three times. He also
has a history of easy bruising and arterial rupture.
• PE: Hyperelastic skin, hyperextensitivity of joints.
• Micropathology: Collagen fibrils of dermis skin
larger than normal and irregular in outline.
• Discussion: Genetic disease, autosomal dominant
or recessive (10 types). Due to point mutation in a
code for glycine and thus disruption of the
collagen triple helix. It can also be due to the exon
skipping which shortens the polypeptide.
Familial Hyperinsulinemia
• Autosomal dominant condition which is
characterized by mild diabetes.
• Approximately equal amounts of insulin
and an abnormally processed proinsulin is
released into the circulation.
• Three enzymes process proinsulin;
endopeptidase that cleaves the Arg31-
Arg32 & Lys64-Arg65 peptide bonds and
carboxypeptidase.
• Defect is due to substitution of Arg65 by
HIS or LEU which prevents the cleavage
between the C-peptide and the A chain of
insulin resulting in secretion of a partially
processed proinsulin.
Glycosylation of Proteins by
ER
Glycosylation of proteins to form
glycoprotein
is important for two reasons:
3. Glycosylation alters the properties of
proteins changing their stability &
physical bulk.
4. Carbohydrates of proteins act as
recognition signals that are central
to aspects of protein targeting and
for cellular recognition of proteins
I-Cell Disease
((Mucolipidosis
• Defects in lysosomal enzyme targeting
due to deficiency of N-acelylglucosamine
phosphate transferase.
• Cells show dense inclusion bodies and the
enzymes are secreted in the medium.
• Patients have high level of lysosomal
enzymes in their sera.
• Characterized by:
– Severe psychomotor retardation
– Skeletal deformaties
– Coarse facial features
– Restricted joint movement
– Death by age of 8
 (Trinucleotide Repeats (1
Case: A thirty-three year old woman has
recently been diagnosed as having mild
myotonic dystrophy (MD). MD is characterized
by autosomal dominant inheritance, muscular
weakness associated with impaired muscular
relaxation, frontal balding, endocrine disorders
such as diabetes and premature ovarian
failure, and premature cataract formation. The
patient has muscular problems and frontal
balding, whereas her affected father, aged 60
years, has only just developed cataracts. She
is concerned about the risk to her own
children if she becomes pregnant.
(Trinucleotide Repeats (2
• The majority of nucleotide repeats are non-coding
(clinically silent), however several are related to
human disease (unstable).
• Unstable repeats undergo changes in size (expansion)
during meiotic division where during each successive
meiosis, expansion of the repeats will gradually
increase in size (anticipation – progressive worsening
of the clinical phenotype with successive meioses).
• Once trinucleotide expansions reach a critical size,
they begin to interfere with gene function and thus
result in the clinical syndrome.
• In the case of MD, with an expansion size of 80
repeats, the patient will exhibit signs of the disease.
• Owing to instability of the triplet repeat during
meiosis, offspring of this patient might expect to have
a larger expansion and also display features of the
disease at a younger age.
• Anticipation may be sex limited, with the largest
expansions only present in eggs, which have larger
amounts of cytoplasm than the spermatozoa
 Protein Truncation Testing in Breast
Cancer
• A hereditary form of breast cancer associated
with coexistence of ovarian cancer has been
shown to be due to mutations in the BRCA1 & 2
gene (tumor suppressor gene).
• Most of the mutations were either nonsense
mutations, altered splice sites, or frameshift
mutations which give rise to abnormally
truncated protein products.
• Screening for mutations using a protein
truncation test has become standard practice
when examining both pathologic tissue and blood
from individuals at risk of possessing a BRCA 1 or
BRCA 2 mutation.
• Comment: The translation of mRNA to the final
protein product results in a peptide of predictable
size. If a gene has a mutation that results in a
premature stop codon, then the resulting peptide
will be abnormally small.
 Reverse transcriptase-PCR
((1

From whole blood, or pathologic tissue,

cDNA complimentary to the mRNA for a
gene can be prepared by the process of
reverse transcription (reverse
transcriptase). RT, using a single poly T
primer, recognizes the mRNA via its poly A
tail.
The resulting cDNA is then used as a
template for a PCR where the entire cDNA
can be amplified to produce a DNA
molecule containing the entire coding
sequence of a gene.
 Reverse transcriptase-
PCR((2
Clearly in some cases, gene expression is
issue-specific and one would not expect
some mRNAs to be present in blood, e.g.
dystrophin from muscle. However, ectopic
transcription of genes occurs in white
blood cells at a low level and allows
analysis of transcripts of genes not
normally expressed.
This can be useful if the desired
transcript is derived from a tissue that is
not easily accessible or if study of the
cDNA can give definite information about
the presence of a mutation.
A Noncompliant Patient (1)

A young man you were treating for a sinus

infection returns to your clinic after 1 week,
still complaining of sinus headaches and
stuffiness. He explains that he began to feel
better about three days after starting to take
the antibiotic tetracycline, which you had
prescribed. You inquire as to whether he
continued to take the full dose of the drug,
even after he began to feel better. He
reluctantly admits that, as soon as he felt
better, he stopped taking the drug. How do
you explain to your patient that it is important
that he takes the drug for as long as you
prescribed it, even if he feels better after only
a few days?
A Noncompliant Patient (2)

Comment: As a physician, you know that

tetracycline inhibits the protein synthetic
machinery of the bacterial cell by binding to the
A site of the ribosome. You also know that, if the
drug is removed, protein synthesis can resume.
If the drug is not taken for the entire period
recommended, bacteria will begin to grow again,
leading to the resurgence of the infection.
Further, those bacteria that begin to grow after
early termination of treatment are likely to be
the most resistant to the drug, either because of
the selection of more resistant strains, or
because of mutation to more resistant strains.
The secondary infection is therefore likely to be
more difficult to control.
(The Case of a Stubborn Microbe (1

A patient you were treating for an infected

wound last week returns to your clinic,
complaining that the infection is worse.
You examine the wound and confirm that
indeed the infection has become worse,
even though you had prescribed a high-
dose regimen of antibiotics that targeted
the bacterial protein synthetic machinery.
After questioning the patient to ensure
that she was compliant and took the
medication, you elect to prescribe
rifampicin, a synthetic derivative of the
naturally occurring antibiotic, rifamycin.
The patient asks you why you think this
antibiotic will work.
(The Case of a Stubborn Microbe (2

Comment: Antibiotics work by targeting

specific functions in the bacterial cell. In
the first round of treatment, you used
antibiotics that inhibited the bacterial
protein synthetic machinery. In some
cases, microbes become resistant to a
specific type of antibiotic and it is
necessary to target other bacterial
functions in order to clear up the
infection. Rifampicin inhibits the trans-
criptional machinery of bacteria,
inhibiting the β subunit of bacterial RNA
polymerase. Without the ability to make
RNA, the bacterial cell will die.
(Therapeutic Application of Ribozymes ( HIV

• They have the potential to become useful

therapeutic agents.
• Inhibitors of viral gene expression such as
HIV using Ribozyme have the following
advantages over other strategies.
– 1. Cleavage of viral transcripts results in direct
irreversible inactivation of the target RNA.
– 2. Few ribozymes may be required to inhibit a
given target gene effectively because a single
ribozyme can catalyze multiple cleavage
reactions and thus destroy multiple viral
transcripts.
(Note – Ribozyme may also be sensitive to HIV
sequence heterogeneity)
(Therapeutic Application of Ribozyme ( Oncogenes

• Neoplastic transformation is often

associated with the expresion of mutant
oncogenes.
• Ribozymes can be designed to inhibit the
expression of specific gene product.
• It has been reported that hammerhead
ribozymes are able to suppress the
tumorigenic properties of cell harboring an
activated human ras gene.
• Lymphcytic leukemia and chronic
myelogenous leukemia express the
transformation of an abnormal mRNA, this
transcript has been shown to be cleared
 Antisense
Oligonucleotide
• Antisense oligonucleotides are short
synthetic olionucleotides (15-25
bases).
• Designed to hybridize to mRNA
through watson-crick base pairing.
• Upon binding to target RNA, the
oligonucleotide prevent expression of
the encoded protein product in a
sequence specific manner.
• Currently one antisense product on
the market used in therapy.
 Suicide Gene
Therapy
• This requires two factors:
1. Introduction of a gene for an enzyme
which is not expressed by the
mammalian cell.

2. A non-toxic pro-drug which can be

converted into a toxic metabolite.

• Most widely used suicide gene is the

herpes simplex virus thymidine
kinase (HSV-tk) gene, used with the
pro-drug Ganciclover.
 HSV-tk Suicide Gene
• This strategy is effective against solid tumors. It is
generally less effective against hematopoietic
malignancies.
• tk enzyme is foreign to mammalian cells and
converts nonactive ganciclovir into a toxic
product (monophosphate form).
• Intracellular host kinases convert the
monophosphate into di and tri phosphate form.
• Triphosphate form is incorporated into the
replicating DNA chain and inhibits DNA
polymerase which results in replication
termination and cell death.
• Bystander effect is also seen, where neighboring
normal cells are also effective due to exchange of
toxic metabolites.
 Disadvantage of Suicide Gene
Therapy
• It requires S-phase cell cycle activity
and thus targets only dividing cells
(not all malignant cells within a tumor are cycling
but different rates of cell doubling is seen and
this requires a longer time of gene expression)
• Although bystander effects help
augment the anti-tumor effects,
there is a likely threshold of HSV-tk
transfer and expression and
persistence of the enzyme which
must be achieved to generate or
sustain a significant therapeutic
effect.
 Cytosinedeaminase
System
• This enzyme is found in fungi and is
not expressed in mammalian cells. It
catalyzes deamination of cytosine into
uracil.
• It can also convert pro-drug 5-
flurocytosine into the metabolite 5-
flurouracil.
• Intracellular enzymes convert 5-
flurouracil into 5-flurouridine-5-
triphosphate and 5-fluro-2-
deoxyuridine-5-phosphate, both of
End Of
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